Ist2 in the Yeast Cortical Endoplasmic Reticulum Promotes Trafficking of the Amino Acid Transporter Bap2 to the Plasma Membrane
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{"title"=>"Ist2 in the yeast cortical endoplasmic reticulum promotes trafficking of the amino acid transporter Bap2 to the plasma membrane", "type"=>"journal", "authors"=>[{"first_name"=>"Wendelin", "last_name"=>"Wolf", "scopus_author_id"=>"55305791100"}, {"first_name"=>"Klaus", "last_name"=>"Meese", "scopus_author_id"=>"7801503494"}, {"first_name"=>"Matthias", "last_name"=>"Seedorf", "scopus_author_id"=>"6602235622"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84896968902", "pui"=>"372704141", "sgr"=>"84896968902", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "issn"=>"19326203", "pmid"=>"24416406", "doi"=>"10.1371/journal.pone.0085418"}, "id"=>"5c656baa-c46e-31be-8833-5e9a289b6b70", "abstract"=>"The equipment of the plasma membrane in Saccharomyces cerevisiae with specific nutrient transporters is highly regulated by transcription, translation and protein trafficking allowing growth in changing environments. The activity of these transporters depends on a H(+) gradient across the plasma membrane generated by the H(+)-ATPase Pma1. We found that the polytopic membrane protein Ist2 in the cortical endoplasmic reticulum (ER) is required for efficient leucine uptake during the transition from fermentation to respiration. Experiments employing tandem fluorescence timer protein tag showed that Ist2 was necessary for efficient trafficking of newly synthesized leucine transporter Bap2 from the ER to the plasma membrane. This finding explains the growth defect of ist2Δ mutants during nutritional challenges and illustrates the important role of physical coupling between cortical ER and plasma membrane.", "link"=>"http://www.mendeley.com/research/ist2-yeast-cortical-endoplasmic-reticulum-promotes-trafficking-amino-acid-transporter-bap2-plasma-me", "reader_count"=>17, "reader_count_by_academic_status"=>{"Researcher"=>5, "Student > Ph. D. Student"=>7, "Student > Master"=>1, "Student > Bachelor"=>2, "Professor"=>2}, "reader_count_by_user_role"=>{"Researcher"=>5, "Student > Ph. D. Student"=>7, "Student > Master"=>1, "Student > Bachelor"=>2, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>11}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>11}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Unspecified"=>{"Unspecified"=>2}}, "reader_count_by_country"=>{"Canada"=>1, "Italy"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1341846"], "description"=>"<p>(A) WT (BY4742 = <i>leu2</i>Δ<i>0, his3</i>Δ<i>1, lys2</i>Δ<i>0, ura3</i>Δ<i>0</i>; closed squares) and <i>ist2</i>Δ (open squares) cells were grown in YPD (grey) or HC (black) media at 30°C. Cells were diluted to 0.1 OD<sub>600</sub> from pre-cultures grown in HC for 24 hours at 25°C. (B) Growth of <i>ist2</i>Δ cells relative to WT ( = 100%) 8 h after dilution into YPD (grey) and HC (black) media. (C) Growth of <i>ist2</i>Δ cells relative to WT ( = 100%) at 25°C (black), 30°C (grey) or 37°C (white) 8 h after dilution into HC medium. (D) WT and <i>ist2</i>Δ cells were pre-cultured for 14 hours in HC medium to 2.60±0.94 and 3.50±0.94 OD<sub>600</sub>, respectively and inoculated to 0.1 OD<sub>600</sub> into fresh HC medium. At the indicated time points the glucose concentration (g/L) of the medium was determined. Additionally, cells were diluted to 0.1 OD<sub>600</sub> into fresh HC medium, grown for 8 hours and relative growth of <i>ist2</i>Δ cells was determined (see cartoon for overview; pc: pre-culture). Growth phase was classified as respiratory (grey) when media glucose concentration was <0.1 g/L. Error bars indicate s.d.m., n = 3. (E) WT (black) and <i>ist2</i>Δ (white) cells were grown in HC media containing 3% glycerol and 2% ethanol as sole carbon source for 24 hours to 2.4±0.7 and 2.4±0.7 OD<sub>600</sub> or for 65 hours to 10.9±0.7 and 11.2±0.3 OD<sub>600</sub>, respectively. Cells were diluted to 0.1 OD<sub>600</sub> into fresh HC media containing 2% glucose or 3% glycerol and 2% ethanol and grown for 8 hours. OD<sub>600</sub> was normalized to the respective WT and is shown as mean+s.d.m. (n = 6 for 24 hours pre-cultures and n = 3 for 65 hours pre-cultures). (F) WT and <i>ist2</i>Δ cells were grown in HC medium with 2% glucose for 18 hours until glucose depletion. Cultures were supplemented with 2% glucose, 2% galactose or mock treated with water for 30 min and diluted to 0.1 OD<sub>600</sub> into HC medium containing 2% glucose and grown for 8 hours. OD<sub>600</sub> was normalized to the respective WT. Error bars indicate s.d.m (n ≥5). The glucose concentration was measured after 30 min of treatment. Triple asterisks indicate significance of p<0.001 (unpaired, two-tailed student’s t-test).</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "cell membrane", "Membrane proteins", "proteins", "Transmembrane proteins", "Transmembrane transport proteins", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "Cell growth", "Membranes and sorting", "fermentation", "respiration", "impaired"], "article_id"=>896743, "categories"=>["Biological Sciences"], "users"=>["Wendelin Wolf", "Klaus Meese", "Matthias Seedorf"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085418.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transition_from_fermentation_to_respiration_is_impaired_in_ist2_/896743", "title"=>"Transition from fermentation to respiration is impaired in <i>ist2</i>Δ.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-08 03:42:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/1341847"], "description"=>"<p>(A) Cartoon illustrating the localization of compartment-specific pH probes. pH-T78C and pH-T8C exclusively localize to the cortical ER while Get2-pH and pH-Get2 localize to the general ER. (B) <i>In vivo</i> measurement of cytosolic pH in cells grown for 12 to 27 hours in HC medium lacking leucine. Growth phases (fermentative, transition and respiratory) are indicated. WT cells expressing pHluorin (black squares) or pH-Get2 (blue triangles) were analyzed by flow cytometry. Error bars indicate s.d.m. (n = 2). (C) pH in the cytosol (black) and general ER lumen (grey) of WT (filled bars) and <i>ist2</i>Δ (open bars) cells expressing pH-Get2 and Get2-pH under fermentative and respiratory conditions. Error bars indicate s.d.m. (n = 4). (D) H<sup>+</sup> gradients (mol/L) between cytosol and lumen of the general (black) or cortical (grey) ER in WT (filled bars) and <i>ist2</i>Δ (open bars) cells under fermentative and respiratory conditions. The H<sup>+</sup> concentrations of general and cortical ER cells were determined in cells expressing Get2-pH and pH-T8C, respectively. Cytosolic H<sup>+</sup> concentrations in the vicinity of general and cortical ER were determined in cells expressing pH-Get2 and pH-T7C, respectively. Error bars indicate s.d.m. (n = 4).</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "cell membrane", "Membrane proteins", "proteins", "Transmembrane proteins", "Transmembrane transport proteins", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "Cell growth", "Membranes and sorting", "gradients", "er", "membrane"], "article_id"=>896744, "categories"=>["Biological Sciences"], "users"=>["Wendelin Wolf", "Klaus Meese", "Matthias Seedorf"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085418.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_H_gradients_across_the_ER_membrane_depend_on_transition_to_respiration_/896744", "title"=>"H<sup>+</sup> gradients across the ER membrane depend on transition to respiration.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-08 03:42:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/1341848"], "description"=>"<p>(A) WT (BY4741), <i>pma1-007</i> and <i>pma1-007 ist2</i>Δ cells were grown to mid log phase and spotted on HC and HC plates buffered to pH 3.0. Cells were spotted in five-fold serial dilutions starting from 2.0 OD<sub>600</sub>. (B) Relative growth of WT (black), <i>pma1-007</i> (grey) and <i>pma1-007 ist2</i>Δ (white) cells. Cells were pre-cultured in HC medium for 18 hours, diluted to 0.1 OD<sub>600</sub> into fresh HC medium buffered to pH 3.0 and grown for 24 hours. OD<sub>600</sub> was normalized to WT. (C) As in B but cells were diluted into unbuffered HC media with a pH of 4.65 and grown for 8 hours. Error bars indicate s.d.m. (n = 9).</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "cell membrane", "Membrane proteins", "proteins", "Transmembrane proteins", "Transmembrane transport proteins", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "Cell growth", "Membranes and sorting", "pma1"], "article_id"=>896745, "categories"=>["Biological Sciences"], "users"=>["Wendelin Wolf", "Klaus Meese", "Matthias Seedorf"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085418.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Impaired_function_of_Pma1_does_not_contribute_to_the_ist2_growth_delay_/896745", "title"=>"Impaired function of Pma1 does not contribute to the <i>ist2</i>Δ growth delay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-08 03:42:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/1341849"], "description"=>"<p>(A) Relative growth of WT (black) and <i>ist2</i>Δ (white) strains transformed with <i>CEN</i> plasmids encoding the <i>LEU2</i>, <i>HIS3</i> or <i>LYS2</i> gene. Pre-cultures grown in HC-leu, HC-his or HC-lys media for 18 hours were diluted to 0.1 OD<sub>600</sub> into fresh HC media and grown for 8 hours. OD<sub>600</sub> of transformed <i>ist2</i>Δ strains were normalized to the respective WT strains. Error bars indicate s.d.m. (n ≥6). (B) Relative growth of WT and <i>ist2</i>Δ strains in HC media with the indicated leucine concentration. Note that the leu concentration of standard HC medium is 0.6 mM. Pre-cultures grown in HC medium for 18 hours were diluted to 0.1 OD<sub>600</sub> into fresh media and grown for 8 hours. OD<sub>600</sub> were normalized to WT grown in standard HC media. Error bars indicate s.d.m. (n = 6). (C) Relative growth of WT and <i>ist2</i>Δ strains transformed with <i>LEU2</i>-encoding <i>CEN</i> plasmids at limiting lysine (30 µM), histidine (10 µM) and tryptophane (20 µM) concentrations. Pre-cultures grown in HC-leu for 18 hours were diluted to 0.1 OD<sub>600</sub> into fresh HC-leu media with the indicated lysine, histidine and tryptophane concentrations. OD<sub>600</sub> were normalized to WT grown in standard HC medium. Error bars indicate s.d.m. (n ≥6). (D) Relative growth of WT, <i>bap2</i>Δ and <i>ist2</i>Δ strains transformed with <i>CEN</i> plasmids encoding <i>BAP2</i> under control of the <i>pADH1</i> (light grey) or <i>pTEF1</i> (dark grey) promoters or empty plasmids p416. Pre-cultures grown in HC-ura for 18 hours were diluted to 0.1 OD<sub>600</sub> into fresh HC media and OD<sub>600</sub> was measured after 10 hours. OD<sub>600</sub> was normalized to WT transformed with empty plasmid. White numbers at the base of dark grey bars indicate fold increase compared to respective strains transformed with empty plasmids. Error bars indicate s.d.m. (n ≥3). (E) <i>CAN1</i> under control of <i>pADH1</i> and <i>pTEF1</i> promoters or empty vectors were expressed in WT and <i>ist2</i>Δ cells from <i>CEN</i> plasmids. Cells were grown to the end of <i>log</i> phase, diluted to 0.1 OD<sub>600</sub> into fresh medium and OD<sub>600</sub> was measured after 8 hours. OD<sub>600</sub> was normalized to WT transformed with empty plasmid (black). Error bars indicate s.d.m. (n = 6). Significance (unpaired, two-tailed student’s t-test) of p<0.01, p<0.005 and p<0.001 is indicated by single, double and triple asterisks; n.s. indicates non-significant difference (p>0.01).</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "cell membrane", "Membrane proteins", "proteins", "Transmembrane proteins", "Transmembrane transport proteins", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "Cell growth", "Membranes and sorting", "uptake", "impaired"], "article_id"=>896746, "categories"=>["Biological Sciences"], "users"=>["Wendelin Wolf", "Klaus Meese", "Matthias Seedorf"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085418.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Leucine_uptake_is_impaired_in_ist2_/896746", "title"=>"Leucine uptake is impaired in <i>ist2</i>Δ.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-08 03:42:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/1341850"], "description"=>"<p>(A and C) Representative green channel (Bap2-GFP) and bright field (BF) images of WT and <i>ist2</i>Δ cells expressing genomically tagged Bap2-GFP. Cells were inoculated to 0.2 OD<sub>600</sub> in HC media and grown for 16 to 20 hours (A). After 24 hours (indicated as 0 h HC+leu) cells were diluted 1∶20 into fresh HC media containing (4 h HC+leu) or lacking (4 h HC-leu) leucine and grown for 4 hours. Scale bars correspond to 2 µm. For quantification of the intensity ratio between peripheral and intracellular fluorescence the mean intensity of a ring-like peripheral area was divided by the mean intensity of the intracellular area (B and D). The quantification of the experiment shown in A includes an additional 24 hours time point (B). The quantification of the experiment shown in C includes additional time points 1 to 3 hours after dilution in HC-leu media (D). Error bars indicate s.d.m. (n = 15). Significance (unpaired, two-tailed student’s t-test) of p<0.01, p<0.005 and p<0.001 is indicated by single, double and triple asterisks; n.s. indicates non-significant difference (p>0.01).</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "cell membrane", "Membrane proteins", "proteins", "Transmembrane proteins", "Transmembrane transport proteins", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "Cell growth", "Membranes and sorting", "trafficking", "bap2", "impaired"], "article_id"=>896747, "categories"=>["Biological Sciences"], "users"=>["Wendelin Wolf", "Klaus Meese", "Matthias Seedorf"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085418.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PM_trafficking_of_Bap2_is_impaired_in_ist2_cells_/896747", "title"=>"PM trafficking of Bap2 is impaired in <i>ist2</i>Δ cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-08 03:42:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/1341851"], "description"=>"<p>(A) Relative growth of WT (black) and <i>ist2</i>Δ (white) cells expressing endogenous Bap2 (no tag) or Bap2-tFT. Cells were grown in HC media for 18 hours, diluted to 0.1 OD<sub>600</sub> into fresh medium and grown for 8 hours. OD<sub>600</sub> was normalized to WT expressing untagged Bap2. Error bars indicate s.d.m. (n = 6 for untagged and n = 15 for tFT-tagged cells). (B) Representative images of Bap2-tFT expressed in WT and <i>ist2</i>Δ cells. Bright field (BF), green channel (sfGFP), red channel (mCherry) and ratiometric images are shown after 18 hours growth in HC media or 4 hours after dilution into fresh HC media. Arrows in sfGFP images indicate perinuclear ER. Scale bars in BF images correspond to 2 µm. (C) mCherry/sfGFP intensity ratios of the periphery in WT and <i>ist2</i>Δ cells and of perinuclear ER in <i>ist2</i>Δ. Error bars indicate s.d.m. (n = 40; 10 cells each from 4 images taken with identical settings. Single cell values correspond to mean intensity of three randomly chosen areas within the respective membrane). (D and E) Distribution of the mCherry/sfGFP intensity ratio for Bap2-tFT in the periphery of WT (filled circles) and <i>ist2</i>Δ (open circles) mother (D) and daughter (E) cells. Error bars indicate s.e.m. (n ≥17). (F) Uptake of <sup>3</sup>H-leucine in WT and <i>ist2Δ</i> cells expressing genomically tagged Bap2-tFT and <i>LEU2</i> from a plasmid. Cells were grown for 18 hours in HC media without leucine followed by dilution into HC media containing leucine.</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "cell membrane", "Membrane proteins", "proteins", "Transmembrane proteins", "Transmembrane transport proteins", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "Cell growth", "Membranes and sorting", "synthesized", "bap2", "delayed"], "article_id"=>896748, "categories"=>["Biological Sciences"], "users"=>["Wendelin Wolf", "Klaus Meese", "Matthias Seedorf"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085418.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ER_exit_of_newly_synthesized_Bap2_is_delayed_in_ist2_cells_/896748", "title"=>"ER exit of newly synthesized Bap2 is delayed in <i>ist2</i>Δ cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-08 03:42:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/1341859", "https://ndownloader.figshare.com/files/1341860", "https://ndownloader.figshare.com/files/1341861", "https://ndownloader.figshare.com/files/1341863", "https://ndownloader.figshare.com/files/1341864"], "description"=>"<div><p>The equipment of the plasma membrane in <i>Saccharomyces cerevisiae</i> with specific nutrient transporters is highly regulated by transcription, translation and protein trafficking allowing growth in changing environments. The activity of these transporters depends on a H<sup>+</sup> gradient across the plasma membrane generated by the H<sup>+</sup>-ATPase Pma1. We found that the polytopic membrane protein Ist2 in the cortical endoplasmic reticulum (ER) is required for efficient leucine uptake during the transition from fermentation to respiration. Experiments employing tandem fluorescence timer protein tag showed that Ist2 was necessary for efficient trafficking of newly synthesized leucine transporter Bap2 from the ER to the plasma membrane. This finding explains the growth defect of <i>ist2</i>Δ mutants during nutritional challenges and illustrates the important role of physical coupling between cortical ER and plasma membrane.</p></div>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "cell membrane", "Membrane proteins", "proteins", "Transmembrane proteins", "Transmembrane transport proteins", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "Cell growth", "Membranes and sorting", "yeast", "cortical", "endoplasmic", "reticulum", "trafficking", "amino", "transporter", "bap2", "plasma"], "article_id"=>896756, "categories"=>["Biological Sciences"], "users"=>["Wendelin Wolf", "Klaus Meese", "Matthias Seedorf"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085418.s001", "https://dx.doi.org/10.1371/journal.pone.0085418.s002", "https://dx.doi.org/10.1371/journal.pone.0085418.s003", "https://dx.doi.org/10.1371/journal.pone.0085418.s004", "https://dx.doi.org/10.1371/journal.pone.0085418.s005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Ist2_in_the_Yeast_Cortical_Endoplasmic_Reticulum_Promotes_Trafficking_of_the_Amino_Acid_Transporter_Bap2_to_the_Plasma_Membrane_/896756", "title"=>"Ist2 in the Yeast Cortical Endoplasmic Reticulum Promotes Trafficking of the Amino Acid Transporter Bap2 to the Plasma Membrane", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-01-08 03:42:36"}

PMC Usage Stats | Further Information

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Relative Metric

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